Eighty percent of lung and 50-80% of esophageal cancers are directly attributable to cigarette smoking. Nearly 70% of malignant pleural mesotheliomas (MPM) arise in smokers or former smokers. Although numerous epigenetic alterations have been identified in tobacco-associated thoracic malignancies, limited information is available regarding the sequence of epigenomic events by which tobacco components establish and maintain the DNA methylation paradox during aerodigestive tract carcinogenesis. Conceivably, identification of epigenomic mechanisms that mediate initiation and progression of thoracic malignancies may hasten the development of novel strategies for treatment and prevention of these neoplasms. The specific aims of this project include: 1. Elucidation of epigenomic alterations mediated by cigarette smoke during aerodigestive tract carcinogenesis. 2. Identification of novel epigenomic targets in thoracic malignancies. Novel in-vitro models and correlative experiments with primary tumor/normal tissue specimens have been utilized to identify epigenomic alterations which contribute to initiation and progression of lung and esophageal cancers and malignant pleural mesotheliomas. A steady stream of manuscripts have been published by our group describing the epigenomic effects of cigarette smoke and other environmental carcinogens in normal aerodigestive tract epithelial cells and thoracic cancer cells in-vitro and in-vivo. Presently, an estimated 10-20% adolescents and young adults in the US smoke hookah, which they perceive to be a safe alternative to cigarettes. As such hookah is an emerging health hazard. Recently, a series of experiments have been conducted to examine the epigenenomic and transcriptomic effects of hookah smoke relative to conventional cigarette smoke in normal and immortalized human respiratory epithelial cells. Briefly, primary normal human small airway epithelial cells (SAEC) from three donors, as well as cdk4/hTERT-immortalized SAEC and human bronchial epithelial cells (HSAEC and HBEC, respectively) were cultured for 5 days (short-term) or 12 months (long term) in normal media with or without cigarette smoke condensate (CSC) or water pipe condensate (WPC). CSC and WPC mediated dose-dependent growth inhibitory effects in cultured respiratory epithelia cells, which coincided with decreased levels of global H4K16Ac and H4K20Me3. CSC and WPC mediated distinct as well as overlapping transcriptome signatures, and pathway modulations that were cell line and dose-dependent. 211 and 329, vs 161 and 261 transcripts were commonly modulated across all five lines by low concentration and high concentration CSC, and low concentration and high concentration WPC, respectively. 61 and 96 transcripts were commonly modulated across all five lines by low concentration CSC and WPC, and high concentration CSC and WPC, respectively. Epiregulin (EREG) encoding a master regulator of EGFR signaling which has been implicated in promotion and progression of lung cancers was up-regulated, whereas Filamin A Interacting Protein 1-Like (FILIP1L) encoding an inhibitor of lung cancer invasion was down-regulated in the five cell lines following CSC and WPC exposures. Induction of EREG and repression of FILIP1L by CSC and WPC coincided with DNA methylation changes within the respective promoters. Given the relatively limited overlap between transcriptome signatures mediated by CSC and WPC in respiratory epithelial cells despite large numbers of cancer-associated transcripts and common pathways affected by these condensates, additional experiments were performed to examine if CSC and WPC preferentially modulated cancer-associated transcriptomes in a histology-specific manner. Briefly, cancer associated transcripts uniquely modulated by CSC or WPC or commonly regulated by CSC and WPC in SAEC or immortalized respiratory epithelial cells were compared to expression levels of these transcripts in 10 NSCLC and 10 SCLC lines relative to SAEC using publicly accessible RNA-seq databases. Notably, transcriptome signatures uniquely associated with CSC or WPC exposures or common to both segregated based on NSCLC vs SCLC histology. Subsets of transcripts that were uniquely up-regulated by either CSC, WPC, or both in SAEC were similarly up-regulated in NSCLC lines but down-regulated in SCLC lines and vice versa, whereas subsets of transcripts that were down-regulated by CSC, WPC or both in SAEC were down-regulated in NSCLC yet up-regulated in SCLC lines and vice versa. A similar phenomenon was observed following analysis of CSC and or WPC transcriptome signatures in HBEC and HSAEC. Further studies are necessary to determine the biologic significance of these provocative findings, and to ascertain if these signatures can prove useful in epidemiologic analyses of lung cancers arising in hookah or cigarette smokers, risk assessment in smokers with no clinical evidence of malignancy, and chemoprevention trials in these individuals. A comprehensive manuscript describing these findings has been submitted for publication. Additional studies have focused on delineation of epigenetic mechanisms contributing to malignant pleural mesotheliomas (MPM), and identification of novel therapeutic targets in these neoplasms. Micro-array, qRT-PCR, immunoblot, and immunohistochemistry experiments demonstrated that expression of ubiquitin-like with plant homeodomain and ring finger domains 1 (UHRF1)- a master regulator of DNA methylation was significantly higher in MPM lines/specimens relative to normal mesothelial cells/ normal pleura. Tissue culture experiments demonstrated that asbestos induced UHRF1 expression in normal mesothelial cells. Analysis of two independent databases demonstrated that UHRF1 up-regulation correlated inversely with overall survival in MPM patients. Knockdown of UHRF1 reversed global hypomethylation and up-regulated tumor suppressor gene expression, inhibiting proliferation, invasion, and clonogenicity of MPM cells, as well as growth of MPM xenografts. Constitutive over-expression of p53 significantly reduced UHRF1 expression in MPM cells. These effects were phenocopied by exposure of MPM cells/xenografts to the repurposed chemotherapeutic agent, mithramycin, which we have previously shown to deplete the oncogenic transcription factor SP1, and up-regulate and activate p53 in MPM cells. These results provide further preclinical rationale for our recently initiated phase I/II evaluation of mithramycin in patients with thoracic malignancies. Collectively, our findings provide the first evidence that UHRF1 is a novel, druggable epigenetic driver in malignant pleural mesotheliomas, and warrant further studies to optimize pharmacologic regimens that inhibit UHRF1 expression/activity for mesothelioma therapy. A manuscript pertaining to these studies has been submitted for publication.